Numerous known patented devices exist in the art of automated sampling of multiple sources for analysis in liquid chromatograph devices.
In early liquid sampling devices, typified in U.S. Pat. No. 4,478,095 to Bradley, et al, and containing samples were disposed around the periphery of a rotatable turntable or carousel.
These devices used a positive displacement system for drawing up a quantity of sample from the vials, wherein a co-axial needle was used to pierce a septum covering the vials, and air or an inert gas injected through an outer annulus within the co-axial needle to force a quantity of sample up through the inner annulus of the needle into a conduit means.
The quantity of sample was then conveyed by the conduit means to an injector valve, and into a sample loop affixed at both its inlet end, and outlet end, to such injector valve. The rotary component of the valve was then rotated to connect one end of the sample loop with a supply of pressurized solvent, and the other end of the sample loop was placed in communication with a conduit leading to a liquid chromatograph column. The quantity of sample was thereby flushed from the sample loop by the pressurized solvent, and thusly flowed into the chromatograph column together with the solvent for analysis.
FIG. 1 of U.S. Pat. No. 4,478,095 to Bradley et al clearly shows such a device, having a conduit 49 leading from the vials to an injector valve 51, and also a syringe means 139 for aiding the withdrawal of the quantity of sample from the vials 27. The quantity of sample is drawn into the sample loop 143 connected to the injector valve 51, and the valve is then switched to connect a pressurized flow of solvent in conduit line 153, which then flushes the quantity of sample into conduit line 155 for transfer to a liquid chromatograph column.
FIGS. 4 and 5 of U.S. Pat. No. 3,918,913 disclose a similar device for withdrawing a quantity of sample from a vial 11, and injecting such quantity into a liquid chromatograph column 6, operating on the identical principal.
The problem with such prior art devices was that a portion of the sampled quantity always lay in the conduit line leading from the sample container to the injection valve, and was never able to be injected into the column for analysis.
Accordingly, only sample volume which lay in the sample loop (line 5 in FIGS. 4 and 5 of Stevenson et al, and line 143 in FIG. 1 of Bradley et al) was able to be injected into the liquid chromatograph column for analysis, and the portion of the sampled volume lying in the conduit line leading to the valve (conduit line 21 in FIGS. 4 and 5 of Stevenson et al, and conduit line 49 of FIG. 1 of Bradley et al) was unusable, and had to be purged from the system by means of flushing solvent, as disclosed in U.S. Pat. No. 4,478,095 to Bradley et al, to avoid contamination with a subsequently drawn quantity of sample from another sample container.
In some applications for sampling devices, it is extremely critical to avoid any loss of the sampled quantity, since as much as possible of the sampled quantity is desired to be injected into the liquid chromatograph for analysis. For example, in hospital and medical use, the quantity of sample contained in vials where the sample is a blood, tissue, or bone marrow specimen obtained from a child, or a small localized area of infection in an individual, may indeed be a very small quantity, often much less than 20-100 microliters.
Accordingly, as much as the sample must be injected as possible into the liquid chromatograph column to obtain as accurate an analysis as possible of the components within the sampled quantity.
In order to overcome the problem of sample loss, it is known in the prior art to incorporate the sampling conduit line as part of the sample loop, whereby all of the sampled quantity can then be injected through the injector valve into the liquid chromatograph column.
U.S. Pat. No. 4,242,909 to Gundelfinger, assigned to Rheodyne Incorporated, discloses such a device. Accordingly, in FIGS. 7 and 8 thereof, an injector valve 156 in a load position allows the valve ports on the injector valve to connect a syringe means 19 with an overfill loop 18 whose loop end 22 is inserted into a sample container 12 to allow the syringe means 19 to withdraw a sample therefrom. The injector valve 156 when switched to an inject position, operatively disconnects the overfill loop 18 with the syringe means 19 and connects one end thereof 44 to a pressurized flow of solvent, and the other end (i.e. the loop end 22) is allowed to be inserted into a loop end coupling 152 in injector valve 154, which is in fluid communication with the liquid chromatograph column 14.
While the Gundelfinger configuration completely overcomes the problem of sample loss, since all of the sampled quantity drawn into the overfill loop 18 is injected into the injector valve 154 and into the liquid chromatograph column 14, and further that by placing the loop end 22 directly into the injector valve 154, certain other advantages discussed below are realized, a definite and important drawback of the Gundelfinger configuration lies in the fact that all of the quantity drawn into the overfill loop 18 must be injected at one time into the injector valve 154 and chromatograph column. Accordingly, because the overfill loop 18 comprises part of the flow line between the high pressure pump 32 and liquid chromatograph column, no capability exists for repetitively injecting aliquot portions of the sampled quantity from the overfill loop 18 to the chromatograph column. This is important, since duplicate or triplicate results from chromatograph testing cannot be obtained, which greatly lessens confidence in the single result thereby obtained.
The Gundelfinger type configuration accordingly presents serious disadvantages then in pharmaceutical or immunilogical testing applications, since frequently the testing regimen specified in laboratory procedures requires repetitive liquid chromatograph testing on the same sampled quantity. Accordingly, for the Gundelfinger configuration to accomplish this result, only a portion of the sample contained within the sample container can be withdrawn at one time for analysis. This requires much greater complexity in the apparatus to accomplish this result, and the applicant herein presently knows of no device using such configuration that has been able to accomplish this result.
Accordingly, a real need exists in the art for a sampling device that is able to utilize all of the sampled quantity for subsequent injection into a liquid chromatograph column without sample loss, and further be able to repetitively inject equal portions of the same sampled quantity into a liquid chromatograph column to thereby improve the reliability in the results obtained from such liquid chromatograph testing.
As mentioned above, the placement of a loop end, or injector tube, directly into an injector valve, wherein the loop end within the valve is then allowed to be in direct fluid communication with the sample loop, avoids the problem that of the earlier valve designs had in that some of the sample would remain in the flow passage of the injector valve between the injector tube and the sample loop, and thus not be injected. This advantage is known in the art, and is clearly disclosed in U.S. Pat. No. 4,182,184 to Bakalyar, also assigned to Rheodyne, which relates to an injector valve specifically incorporating such feature.
Such patent discloses a manual method, however, for use of such injector valve wherein a micro-syringe 52, as shown in FIG. 6 thereof, is inserted into the injector valve 50 to inject an entire previously sampled quantity into a sample loop 72, and the rotor 58 of the injector valve 50 is rotated, as shown in FIG. 8 thereof, to connect the sample loop 72 at one end with a pressurized source of solvent, and at the other end with a liquid chromatograph column, to flush the sample into the liquid chromatograph column.
The Bakalyar patent does not, however, teach or disclose the method of injecting aliquot portions down to 1-10 microliters in volume from a single sampled quantity of sample previously drawn into the micro syringe, and then switching the injector valve 50 from a load position to an inject position and back to a load position, to repetitively inject portions of such sampled quantity into the liquid chromatograph column. In fact, such patent clearly discloses at page 3, lines 65-68 thereof that the sample loop is always made long enough to contain a volume of liquid greater than the largest volume to be introduced by the micro syringe 52.
To achieve the ability to repetitively inject microliter portions of a sampled quantity into the injection valve from a syringe means, automation of the injection valve and syringe means are required, since manual sensitivity is not adequate to dispense such small volumes from the micro syringe into the injection valve.
It was not immediately apparent then from the prior art how the Bakalyar injection valve with its above advantages may be automated. This was due to the fact that in most applications the syringe needle passageway 104 was contemplated as being located in the rotor, offset from the axis of rotation thereof, and accordingly rotation of the rotor moved the syringe needle passageway making it difficult for automatic means to align the syringe needle with the syringe passageway in the valve.
U.S. Pat. No. 4,242,909 to Gundelfinger, a later patent also assigned to Rheodyne Incorporated does disclose in FIGS. 7 and 8 thereof an automated sampler-injector design utilizing the Bakalyar valve. However, as discussed earlier, such configuration only allowed the entire contents of the overfill loop 18 to be injected into the injector valve 156, and accordingly aliquot portions of the sampled quantity were unable to be dispensed into the valve. Moreover, such patent clearly disclosed on page 6, lines 61-68 and page 7, lines 1-13 that the tube receiving passage 152 located in the rotor was to be rotated. Accordingly, since the loop end 22 can only be inserted into the loop end receiving passage 152 when such passage is aligned with the liquid chromatograph column 14, should the column be under pressure, it is possible leakage of the solvent from the column 14 could occur from the loop-end receiving passage 152, and also leakage of the sample contained in the overfill loop 22 from the loop end 22, up to the point in time the loop end 22 is lowered into the loop-end passage 152 for injection of the sample into the valve 156. Accordingly, it is unclear from Gundelfinger precisely how the Bakalyar valve is to function effectively within the parameters of the design disclosed in Gundelfinger.